WO2000043782A2 - Elimination des prions du sang, du plasma et d'autres liquides - Google Patents

Elimination des prions du sang, du plasma et d'autres liquides Download PDF

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Publication number
WO2000043782A2
WO2000043782A2 PCT/US1999/030167 US9930167W WO0043782A2 WO 2000043782 A2 WO2000043782 A2 WO 2000043782A2 US 9930167 W US9930167 W US 9930167W WO 0043782 A2 WO0043782 A2 WO 0043782A2
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Prior art keywords
prp
complexing agent
prion
sample
prions
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PCT/US1999/030167
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WO2000043782A3 (fr
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Stanley B. Prusiner
Jiri G. Safar
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The Regents Of The University Of California
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Priority to KR1020017009089A priority Critical patent/KR20010089612A/ko
Priority to AU25898/00A priority patent/AU768032B2/en
Priority to IL14409799A priority patent/IL144097A0/xx
Priority to CA002363846A priority patent/CA2363846A1/fr
Priority to BR9916932-0A priority patent/BR9916932A/pt
Priority to JP2000595152A priority patent/JP2002539081A/ja
Priority to AT99968494T priority patent/ATE299268T1/de
Priority to DE69926081T priority patent/DE69926081T2/de
Priority to EP99968494A priority patent/EP1145013B1/fr
Publication of WO2000043782A2 publication Critical patent/WO2000043782A2/fr
Publication of WO2000043782A3 publication Critical patent/WO2000043782A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3618—Magnetic separation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/07—Proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to methods of purifying samples and particularly to methods of removing prions from blood and blood products.
  • Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
  • PrP prion protein
  • Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown et al., Lancet 340:24- 27 (1992)]. Despite numerous attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date [Harries- Jones et al., J. Neurol. Neurosurg. Psychiatry 57:1113-1119 (1988)] except in cases of iatrogenically induced disease.
  • mice expressing the human, or ovine PrP transgenes When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long. Thus, it has not been possible, for example in the case of human prions, to use transgenic animals (such as mice containing a PrP gene of another species) to reliably test a sample to determine if that sample is infected with prions. The seriousness of the health risk resulting from the lack of such a test is exemplified below.
  • HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH [Gibbs, Jr. et al., N. Engl. J. Med. 325:358-359 (1993)].
  • Prions are removed from biological materials such as blood and plasma by contacting these materials with a complexing agent, such as a biological agent (e g an antibody) or a chemical agent (e.g a substrate comprised of sodium phosphotungstate) which binds prions
  • a complexing agent such as a biological agent (e g an antibody) or a chemical agent (e.g a substrate comprised of sodium phosphotungstate) which binds prions
  • a complexing agent such as a biological agent (e g an antibody) or a chemical agent (e.g a substrate comprised of sodium phosphotungstate) which binds prions
  • a complexing agent such as a biological agent (e g an antibody) or a chemical agent (e.g a substrate comprised of sodium phosphotungstate) which binds prions
  • the substrate may be used in any configuration which allows removal of the prion from the biological materials, such as spherical polymer beads which are coated with a complexing agent and housed in a column
  • One embodiment of the invention is a method of removing p ⁇ ons from a sample such as human blood by contacting the sample with a substrate comprised of a prion complexing agent such as a sodium salt of phosphotungstic acid and allowing prions in the blood to bind to the complexing agent for removal
  • a device which removes prions from a liquid sample which device is comprised of substrate such as ferromagnetic beads coated with a polymer with a complexing agent thereon wherein the substrate is preferably positioned in a tubular housing through which liquid sample flows.
  • An object of the invention is to provide a simple and economical means for removing prions from a material.
  • An advantage of the invention is that blood products which might contain prions can be certified as prion free when processed via the invention.
  • a feature of the invention is that prions selectively bind to chemical agents such as heteropoly acids or metal salts of heteropoly acids.
  • a preferred chemical agent for use in the methods of the invention is sodium phosphotungstate.
  • prions bind selectively to biological agents such as peptides, small molecules and selective PrP Sc binding antibodies.
  • An aspect of the invention is a substrate which may be in the form of spherical polymer beads having a complexing agent coated on their surface.
  • spherical beads have a ferromagnetic metal core which allows separation of the beads using magnetic forces and have an inert polymer coating the metal core with the polymer coated with a metal salt of phosphotungstate acid.
  • Another aspect of the invention is blood and blood products such as plasma which have been treated with the method of the invention.
  • Yet another aspect of the invention is a filter membrane used to remove prions from biological materials using methods such as hemofiltration.
  • Yet another aspect of the invention is a device comprised of a housing (preferably cylindrical) having surfaces therein coated with a composition which binds PrP Sc e.g. spherical lead surfaces coated with sodium phosphotungstate.
  • complexing agent is used herein to refer to any material which binds or complexes selectively with either the constrictive conformation of a protein (e.g. with PrP Sc ) and/or with the relaxed conformation of a protein (e.g. PrP c ).
  • This complexing agent may be a biological molecule such as a peptide or antibody, e.g. an antibody selective for PrP Sc , or a chemical agent, e.g. phosphotungstic acid (PTA), which may be added in the form of a salt, e.g. sodium phosphotungstate.
  • PTA phosphotungstic acid
  • the complexing agents may be used singly or in combination.
  • a biological complexing agent may be used in tandem with a chemical complexing agent, such as the use of a peptide and a chemical agent.
  • two complexing agents of the same class can be used together, e.g. a mixture of phosphotungstic acid (and salts thereof) and trichloroacetic acid.
  • the complex formed must provide some means for separating the complex from the remainder of the composition, such as immobilization of the complexing agent to a surface.
  • a preferred complexing agent which binds PrP Sc more readily than it binds PrP c and a particularly preferred agent binds PrP Sc with a high degree of affinity and does not bind PrP c at significant levels.
  • a preferred binding agent binds PrP Sc with twice or more the binding affinity as it binds PrP c and preferably five times or more the binding affinity as it binds PrP c .
  • protein as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • the term includes naturally occurring proteins and peptides as well as those which are recombinantly or synthetically synthesized.
  • the term "protein” is specifically intended to cover naturally occurring proteins which occur in at least two different conformations wherein both conformations have the same or substantially the same amino acid sequence but have two or more different three dimensional structures.
  • the two conformations of the protein include at least one conformation which is not related to a disease state and at least one conformation which is related to a disease state — pathogenic.
  • a specific and preferred example of a protein as used in connection with this disclosure is a PrP protein which includes the non-disease form referred to as the PrP c form and the disease related form referred as the PrP Sc .
  • a pathogenic protein as used in connection with this disclosure is not necessarily a protein which is the specific causative agent of a disease.
  • PrP protein refers to both the infectious particle form PrP Sc known to cause diseases (spongiform encephalopathies) in humans and animals and the noninfectious form PrP c which, under appropriate conditions is converted to the infectious PrP Sc form.
  • Particles are comprised largely, if not exclusively, of PrP Sc molecules encoded by a PrP gene. Prions are generally PrP Sc dimers. Prions are distinct from bacteria, viruses and viroids. Known prions infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats, as well as bovine spongiform encephalopathy (BSE), or "mad cow disease", and feline spongiform encephalopathy of cats.
  • BSE bovine spongiform encephalopathy
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Schemker Disease (GSS), and (4) fatal familial insomnia (FFI).
  • CJD Creutzfeldt-Jakob Disease
  • GSS Gerstmann-Straussler-Schemker Disease
  • FFI fatal familial insomnia
  • PrP gene is used herein to describe genetic material which expresses proteins including known polymorphisms and pathogenic mutations.
  • PrP gene refers generally to any gene of any species which encodes any form of a PrP protein. Some commonly known PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992), and U.S. Patents 5,565,186; 5,763,740; 5,792,901; and WO97/04814, incorporated herein by reference to disclose and describe such sequences.
  • the PrP gene can be from any animal, including the "host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • the protein expressed by such a gene can assume either a PrP c (non-disease) or PrP Sc (disease) form.
  • antibody stands for an immunoglobulin protein which is capable of binding an antigen.
  • Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g. F(ab)', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • Preferred antibodies for assays of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to a protein of interest e.g., a PrP protein and specifically a PrP Sc protein or PrP Sc dimer.
  • Antibodies which are immunoreactive and immunospecific for both the native non-disease form and disease form may be used.
  • Antibodies for PrP are preferably immunospecific - e.g., not substantially cross-reactive with related materials. Some specific antibodies which can be used in connection with the invention are disclosed in published PCT application WO 97/10505 which is incorporated herein by reference to disclose and describe antibodies. This published PCT application corresponds to U.S. Patent 5,846,533 issued December 8, 1998 also incorporated herein by reference.
  • the term "antibody” encompasses all types of antibodies, e.g. polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for both native PrP c and PrP Sc and those with greater binding affinity for PrP Sc are preferred.
  • An antibody of the invention is a "complexing agent" as defined herein.
  • An antibody for binding to PrP c is the monoclonal antibody 263K 3F4 produced by the hybridoma cell line ATCC HB9222 deposited on October 8, 1986 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and disclosed and described in U.S. Patent 4,806,627 issued February 21, 1989 - incorporated by reference to disclose antibodies which selectively bind PrP ⁇ but not PrP Sc
  • “Purified antibody” refers to that which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody
  • a purified antibody of the invention is preferably immunoreactive with and immunospecific for a specific species and more preferably immunospecific for native PrP Sc .
  • Antigenic fragment of a protein (e.g., a PrP protein) is meant a portion of such a protein which is capable of binding an antibody.
  • binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide e.g., epitope of a protein, e.g., a PrP Sc .
  • Antibody binding to its epitope on this specific polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest e.g., binds more strongly to epitope fragments of a protein such as PrP Sc so that by adjusting binding conditions the antibody binds almost exclusively to an epitope site or fragments of a desired protein such as an epitope fragment of PrP Sc .
  • detectably labeled antibody By “detectably labeled antibody”, “detectably labeled anti-PrP” or “detectably labeled anti-PrP fragment” is meant an antibody (or antibody fragment which retains binding specificity), having an attached detectable label.
  • the detectable label is normally attached by chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art.
  • Detectable labels known in the art, but normally are radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
  • detectable label/substrate pairs e.g., horseradish peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin
  • methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).
  • Europium is a particularly preferred label.
  • CNS central nervous system
  • GdnHCl for Guanidine hydrochloride
  • GSS for Gerstamnn-Strassler-Scheinker Disease
  • MoPrP for mouse prion protein
  • SHa for a Syrian hamster
  • PrP Sc for the scrapie isoform of the prion protein
  • PrP c for the cellular contained common, normal isoform of the prion protein; PrPCiDfor the CJD isoform 0 f a PrP pro t e in; FVB for a standard inbred strain of mice often used in the production of transgenic mice since eggs of FVB mice are relatively large and tolerate microinjection of exogenous DNA relatively well;
  • PrP protein may assume its cellular form, i.e. PrP c form or its scrapies form, i.e. PrP Sc form.
  • PrP c cellular form
  • PrP Sc scrapies form
  • One form of the protein is harmless (e.g. PrP c )
  • another form of the protein is pathogenic (e.g. PrP Sc ).
  • PrP Sc pathogenic form of the protein such as PrP Sc
  • the constricted, pathogenic form of the protein such as PrP Sc is present in an animal in very small amounts the animal is not showing symptoms of disease. However, the animal will develop a disease related to the pathogenic form of the protein - e.g. develop a prion disease.
  • the present invention is useful with respect to (1) eliminating the pathogenic form of the protein from the sample such that the material is rendered “prion free” and/or (2) reducing the concentration of the pathogenic form of the protein in a material to a level such that the material is rendered "non-infectious.”
  • the present invention makes it possible to remove prions from a biological sample by exposing the sample to a complexing agent, which binds (preferably selectively) to PrP Sc and allows removal of the PrP Sc
  • a complexing agent which binds (preferably selectively) to PrP Sc and allows removal of the PrP Sc
  • the present process is especially, though not exclusively, useful for the treating of and/or removal of PrP Sc from whole blood. Removal may be through complexing with an immobilized complexing agent, i.e. exposure of the sample to an affinity column, membrane, filter, or beads with immobilized complexing agent.
  • the complexing agent will effectively remove the PrP Sc from the sample, while allowing the sample to stay substantially in the same form to allow proper use of the sample, i.e. maintaining proper pH, structural integrity of cells and proteins, and the like.
  • any type of sample can be processed using the present invention in order to remove a pathogenic form of a protein.
  • the invention could be applied to removal of a constricted form of any protein having a constricted and relaxed form, the invention is described specifically with respect to removal of the pathogenic form of a PrP protein, i.e. concentrating and removing PrP Sc .
  • a biological sample to be treated should be in a liquid flowable form at room temperature (15°C to 30°C).
  • the solution should have a pH of about 6.4 to 8.4, preferably
  • the next step is the most important in the process of the invention.
  • a sample is exposed to a complexing agent which is immobilized on a solid surface or otherwise provided in a manner allowing separation of the prion-bound complexing agent from the sample.
  • the complexing agent forms a complex with or somehow binds preferentially with or exclusively to any constricted (generally a pathogenic form) of the protein present in the sample, thus effectively immobilizing any PrP Sc present in the sample to the solid surface upon exposure of the sample to the immobilized complexing agent.
  • a chemical agent such as a heteropoly acid (e.g. PTA), or preferably a metallic salt thereof (NaPTA) is immobilized to a solid surface such as a membrane filter, a magnetic bead, and the like.
  • PTA heteropoly acid
  • NaPTA metallic salt thereof
  • the sample is subjected to a the complexing agent over a period of time sufficient to allow substantially all the PrP Sc in the sample to complex with the PTA.
  • the sample could be incubated at about 30 °C to 45 °C (preferably 37 °C) over a period of from about 1 to 16 hours.
  • the complexing agent forms a complex with the PrP Sc . What is important is that complex formed can be separated away from the rest of the sample by some means, e.g. filtration, use of magnetic field, sedimentation and the like.
  • the process of the invention produces a biological sample wherein the PrP Sc or other pathogenic protein is substantially removed from the sample, and preferably to levels at which the PrP Sc is undetectable by conventional means. Methods of removal of the PrP Sc will prevent trransmission of PrP Sc -mediated disorders by providing biological samples which are free from infectious levels of prions, i.e. "prion free.”
  • COMPLEXING AGENTS Compounds which are useful as complexing agents in the present invention include antibodies, enzymes, peptides, chemical species, binding molecules, etc. These complexing agents are used in a manner that allows binding and removal of prions from a biologial solution, while maintaining the essential elements of the biological material intact, e.g. retention of cellular morphology and protein integrity. Such complexing agents may be used in whole blood, in blood components such as plasma and platelets, and in other biological fluids as will be apparent to one skilled in the art.
  • the compound for removal of prions from a biological material is a chemical agent that precipitates PrP Sc .
  • One preferred class of chemical agents for use as complexing agents in the present invention are heteropoly acids and salts thereof.
  • Heteropoly acids are fully or partially protonated forms of oxyanions having at least one central element and at least one coordinating element.
  • Heteropoly acids may have the Keggin or Dawson structures.
  • heteropoly acids is the protonated form of heteropolymolybdates. These anions contain from 2 to 18 hexavalent molybdenum atoms around one or more central atoms. About 36 different elements have been identified as central atoms of these heteropolymolybdates. These anions are all highly oxygenated. Examples of heteropolymolybdates include [PMo 12 O 40 ] 3 , [As 2 Mo 18 O 62 , and [TeMo 6 O 24 ] 6 , where the central atoms are P 5+ , As 5+ , and Te 6+ , respectively.
  • heteropolymolybdates A more detailed discussion of heteropolymolybdates is provided in the Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed., 15, 688-689 (1981).
  • Another class of heteropoly acids which is analogous to the protonated form of heteropolymolybdates, is the protonated form of heteropolytungstates.
  • the coordinating element is tungsten instead of molybdenum.
  • the central elements of these heteropoly acids may be selected from the group consisting of P, Si, B, Ge, As, Se, Ti, Zr, Mn, F, V, Ce, and Th.
  • the coordinating elements of these heteropoly acids include Mo and/or W.
  • Optional coordinating elements include V, Mn, Co, Ni, Cu, Zn, and Fe.
  • the ratio of the number of the coordinating elements to the number of central elements may be from 2.5 to 12, preferably from 9 to 12.
  • Particular heteropoly acids which are exemplified in U.S. Pat. No.
  • 4,376,219 include phosphotungstic acid, silicotungstic acid, 10-tungsto-2-vanadophosphoric acid, 6-tungsto-6-molybdophosphoric acid, phosphomolybdic acid, silicomolybdic acid, germanotungstic acid, tungstofluoric acid, and 18-tungsto-2-phosphoric acid as well as salts of all or any of these acids e.g. metal salts such as Na, K, Mg, and Ca salts.
  • a particular heteropoly acid for use in the present invention is phosphotungstic acid, i.e., H 3 PW 12 O 40 and its metal salts particularly Na salts. Such complexing agents effectively bind to PrP Sc .
  • Heteropoly acids of the invention are preferably, although not exclusively, used in a metallic salt form.
  • the metallic salt includes, but is not limited to, sodium, potassium, calcium and the like.
  • the amount of heteropoly acid or salt thereof which is combined with the present support material should be present in an amount sufficient to significantly remove PrP Sc from the a biological fluid, and preferably in an amount sufficient to remove PrP Sc to undetectable levels or at least non-infectious levels.
  • the weight ratio of heteropoly acid to support material may be, for example, from about 1 :20 to about 1 :1.
  • the heteropoly acid may be combined with the support material in any manner which provides adequate dispersion of the heteropoly acid, thereby increasing the effective surface area of the heteropoly acid. A preferred technique for combining these components is by impregnation of the support material with the heteropoly acid.
  • the heteropoly acid may also be combined with the support material by an ion exchange technique.
  • the impregnation technique may involve sorbing an aqueous solution of the heteropoly acid into the porous region of the support material followed by drying to remove water and to leave behind supported heteropoly acid.
  • Other methods of immobilizing heteropoly acids or salts thereof may be used to immobilize these complexing agents, as will be apparent to one skilled in the art upon reading this disclosure.
  • the complexing agent is a protein, peptide, or other biological moeity that selectively binds to PrP Sc .
  • the complexing agents are peptides or other small molecules designed to selectively bind to prions.
  • the peptides or small molecules are designed to preferentially bind to PrP Sc .
  • preferentially bind is meant that the peptide is designed to be at least 20 times or more, more preferably 50 times or more, more preferably 100 times or more, and even more preferably 1000 times or more likely to bind to PrP Sc than to other proteins in the biological solution.
  • Peptides of the invention are preferably designed to bind to the native form of PrP Sc , as opposed to the denatured form, since the biological fluids generally contain PrP Sc in native form. Peptides may be designed to maximize binding to PrP Sc by designing the peptides to areas of PrP Sc that are more accessible to binding, as can be predicted by one skilled in the art.
  • Useful antibodies which bind PrP Sc are disclosed and described in U.S. Patent 5,846,533 issued December 8, 1998 incorporated herein to disclose and describe antibodies and methods of making antibodies. Portions of these antibodies which bind to PrP Sc are peptides which can be bound to a support surface and used in the present invention.
  • peptides may be designed to bind selectively to PrP c or to both PrP Sc and PrP c .
  • PrP Sc form of the prion is the infectious portion, removal of the normal cellular prion proteins can also effectively halt or slow the progression of a prion- mediated disorder.
  • the complexing agent of the invention may also be an antibody selective for prions.
  • This antibody may be directly immobilized or may be bound to another component (e.g. a high density metal). That antibody may bind to PrP Sc , e.g. the antibody disclosed in U.S. Patent 5,846,533.
  • an antibody which binds selectively or exclusively to PrP c may be used.
  • Such an antibody is disclosed in US 4,806,627, issued February 21, 1989, disclosing monoclonal antibody 263K 3F4, produced by cell line ATCC HB9222 deposited on October 8, 1986, which is incorporated herein by reference.
  • the cell line producing the antibody can be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.
  • an indication that no binding occurs means that the equilibrium or affinity constant K a is 10 6 1/mole or less. Further, binding will be recognized as existing when the K a is at 10 7 1 mole or greater, preferably 10 8 1/mole or greater.
  • the binding affinity of 10 7 1/mole or more may be due to (1) a single monoclonal antibody (i.e., large numbers of one kind of antibodies) or (2) a plurality of different monoclonal antibodies (e.g., large numbers of each of five different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations of (1) - (3).
  • Selected preferred antibodies will bind at least 4-fold more avidly to the treated or denatured PrP Sc forms of the protein when compared with their binding to the native conformation of PrP Sc .
  • the four fold differential in binding affinity may be accomplished by using several different antibodies as per (1) - (3) above and as such some of the antibodies in a mixture could have less than a four fold difference.
  • a variety of different methods may be used with one or more different antibodies.
  • antibodies may be labeled with known labels and used with currently available robotics, sandwich assays, electronic detectors, flow cytometry, and the like. Further, the antibodies may be bound to denser components directly or via other intermediates such as anti-antibodies.
  • the complexing agent of the invention may be used in a variety of purification procedures to effectively remove prions from a biological material.
  • a number of methods for use in the present invention are summarized as follows.
  • Affinity Chromatography relies on the interaction of the protein with an immobilized ligand. AC is predicated, in part, on the interaction of ligands attached to chromatographic supports. A hydrophobic ligand coupled to a matrix is variously referred to herein as an AC support, AC gel or AC column. It is further appreciated that the strength of the interaction between the protein and the AC support is not only a function of the proportion of non-polar to polar surfaces on the protein but by the distribution of the non-polar surfaces as well.
  • a number of matrices may be employed in the preparation of AC columns.
  • such matrices are beads, and more preferably spherical beads, which serve as a support surface for the complexing agent of the invention.
  • Suggested materials for the matrices include agarose, cross linked dextran, polyhydroxyl ethyl methacrylate, polyacrylamide, cellulose, and derivatives or combinations thereof, preferably in the form of porous spheres.
  • Cellulose acetate has previously been successfully used in devices for purification of biolological fluids, e.g. extracorporeal blood purification devices.
  • Polyurethane is particularly blood compatible.
  • Silica and its derivatives are also especially useful as support material for use with heteropoly acids. See U.S. Pat Nos. 5,475,178 and 5,366,945, which are incorporated herein by reference.
  • the preferred material for use in the methods of the present invention is agarose, a naturally occurring hydrophilic polymer.
  • a beaded gel with a porosity of from 90-96% is formed by varying the percentage of agarose.
  • the molecular weight of the gel ranges from 0.5 million for 10% agarose to 20 million for 4% agarose. Particle diameters ranging from 20 to 200 microns are commercially available.
  • the mechanical strength of agarose beads can be increased by either increasing the percentage of agarose or crosslinking the beads with epichlorohydrin or 2,3 dibromopropanol, using the method of J. Porath et al. in J. Chromat 60, 167 (1971). This allows a corresponding increase in the maximum operating pressure (a fifty percent increase in agarose leads to a two to four fold increase in the maximum operating pressure).
  • the criteria to determine the appropriate coupling method are: minimization of leakage of the complexing agent from the support, maintenance of the thermal stability of the compound, and retention of the optimum amount of complexing agent.
  • the technique must also not cause a deterioration in the support material or the production of reactive groups on the support which would bind blood components in vivo.
  • the complexing agent must also retain its activity over time.
  • the AC composition of the present invention can be contained within a filtration cartridge for easy use of the composition in a biological fluid purification process.
  • the column composition When the column composition is contained within a single cartridge, it can easily and conveniently be replaced when the purifying capacity of the composition becomes exhausted.
  • the cartridge may be an integral part of a purifying device, in which case the entire device is replaced once the filtration composition has exhausted its efficacy.
  • the support particles with the complexing agent are placed within the cartridge, and the solution to be reacted with the complexing agent is then circulated through the cartridge.
  • Commercially available units for dialysis, blood exchange or oxygenation can be adapted for use as the purifying device.
  • the membrane may have the prion complexing agent conjugated directly to the membrane, either on the side facing the biological fluid or more preferably on the side away from the biological fluid.
  • the complexing agent may be compartmentalized in an area behind the membrane which is inaccessible to the larger components of the biological materials, e.g. blood cells.
  • the complexing agent can be bound to an insoluble matrix behind the membrane.
  • the membrane for use in the present invention may be in planar form, in the form of one or more hollow fibers, and/or in the form of flat foils. See U.S. Pat No. 4,361,484, which is incorporated herein by reference.
  • Suitable materials for the membrane include regenerated cellulose, cellulose acetate, non-woven acrylic copolymer, polysulphone, polyether sulphone, polyacrylonitrile, polyamide and the like.
  • the biologically active material is immobilized in the pores and/or on the surface of the side of the membrane that faces away from the biological fluid. Thereby the components such as blood corpuscles are prevented from contacting the active material.
  • the pores of the membrane are usually of the magnitude of order of 0.01 to 0.8 microns, preferably 0.15 to 0.45 microns, the polymer support must be stable under the conditions of its planned use, i.e. it should not be chemically or enzymatically degraded by blood, the support and immobilized complexing agent must be blood compatible, and the support should have good flow characteristics and low compressibility under clinical flow rates in the range of 150-250 ml/min.
  • the biological fluid need not be exposed to any following filtering for removing possible remaining harmful residues.
  • the separation as the removal of the substances can thereby be performed in one and the same step.
  • the microporous semipermeable membrane can be in the form of individual fibers which are bundled and encapsulated within one and the same casing, with an inlet and outlet for the biological fluid.
  • the ends of the fibers are glued by means of a suitable binder to retain the individual fibers essentially parallel within the casing.
  • One end of the fibers or bundles of fibers is provided in communication with the inlet, while the opposite end is provided in communication with the outlet.
  • the biological material is pumped into the casing through the inlet and through the longitudinal void of the fibers and out of the casing through the outlet.
  • the fluid is exposed to the pressure variations, such that only a penetrating fraction is caused to flow in an alternating path through the fiber walls in each direction for contacting with the prion complexing material.
  • the means for the realization of the pressure variations may again be made up of an expansion chamber in communication with the space between the individual fibers and bundles of fibers, respectively. Any subsequent filtering of the biological material for the removal of possible harmful residues is not needed, since the filtering is automatically achieved through the passage of the fluid through the fiber walls.
  • the pressure variations may vary from -200 to +200 mmHg, preferably from -100 to +100 mmHg.
  • the frequency of the pressure variations may vary from about 0.05 up to about 10 Hz, preferably 0.5 to 1 Hz.
  • the complexing agent is an antibody
  • This general arrangement is preferred when the molecular weight of the antigen is large, e.g., 100,000 Daltons or higher in molecular weight.
  • a six- or eight-carbon methylene group is convenient as a spacer or "handle" between antibody and membrane surface.
  • the spacer molecule may be a protein such a albumin.
  • the outer surface of a membrane can be considered a relatively porous material compared to that of the interior surface which is normally the effective filter surface of an ultrafilter membrane of the asymmetric, sometimes called anisotropic, type.
  • the exterior, porous side of a membrane may be treated with a 17% human albumin solution in saline.
  • the albumin will coat the surfaces within the porous zone of the membrane structure (i.e. the zone that underlies the barrier layer of the membrane) and, thereafter, a solution of protein (e.g. a PrP Sc antibody) can be deposited upon the albumin.
  • a solution of protein e.g. a PrP Sc antibody
  • it is desirable to crosslink the protein somewhat as with a dilute glutaraldehyde solution or some other such mild crosslink-inducing agent); this aids in anchoring the material in place on the membrane surface.
  • One approach to preparing a cartridge which is capable of removing pathogenic factors from blood is an extracorporeal circulation system with fiber membranes having sufficient permeability for the pathogenic blood factor to be removed through the membrane and into a soluble, immobilized antibody sequestered in the extrafiber space.
  • the molecular weight of the immunoreactive complexing agent may be increased to such a size that it will not diffuse, from the exterior, porous, portion of the fiber and into the blood to be purified.
  • the membrane may be composed of two membrane halves which are mechanically generally identical to each other but which chemically may be built up of different material. In this case, it is enough if only the membrane half that faces away from the biological material is able to bind to the prion complexing agent.
  • the membrane halves may be provided in an abutting relationship to each other, wherein the PrP Sc complexing agent preferably is bound in the pores and on both surfaces of the membrane half that faces away from the biological material.
  • the complexing agent e.g. NaPTA or anti-PrP Sc antibodies
  • the prion complexing agent may be bound to an unsoluble matrix behind the membrane.
  • the treating process is yet similar, but since the necessary diffusion distance is about 10 times longer, it may be necessary to arrange a somewhat more real flow through the membrane.
  • the immobilizing procedure is preferably performed such that the complex of prions and the complexing agent remains bound and immobilized, i.e. it is not present in the blood following the purification technique.
  • covalent coupling is the safest immobilization. The nature of covalent coupling used depends on the choice of membrane material and the nature of the complexing agent.
  • Prions can also be removed from biological materials using magnetic partcles comprised of prion complexing agent.
  • the principle components of the magnetic particles of the present invention are a magnetic core.
  • the core consists of particles of iron oxide or other magnetic materials.
  • the PrPSc binding agent of the invention can be incorporated directly on the magnetic core, or indirectly incorporated onto the magnetic core, e.g. through the use of a fibrous material and a binding agent.
  • the fibrous material may comprise an organic polymer in the form of fibers, such as carbohydrate polymers, urea formaldehyde or polynonamethylene urea, and, in particular, cellulose fibers.
  • the binding agent is a material which is introduced between the magentic core and the fiber strands as a liquid, or in solution, and is solidified during the production process of freezing, polymerization or evaporation of a solvent.
  • suitable binding agents are agar, gelatin, an epoxy resin or urea formaldehyde furfuryl alcohol.
  • Magnetic microparticles useful in the present method can be a variety of shapes, which can be regular or irregular; preferably the shape maximizes the surface areas of the microparticles.
  • the magnetic microparticles should be of such a size that their separation from solution, for example by filtration or magnetic separation, is not difficult.
  • the magnetic microparticles should not be so large that surface area is minimized or that they are not suitable for microscale operations. Suitable sizes range from about 0.1. mu. mean diameter to about lOO.mu. mean diameter. A preferred size is about 1.0. mu. mean diameter.
  • Suitable magnetic microparticles are commercially available from PerSeptive Diagnostics and are referred to as BioMag COOH (Catalog Number 8-4125).
  • the coated magnetic particles of the present invention can be produced by stirring or mixing the core particles in a suspension comprising a fibrous material, a prion complexing agent, and a binding agent.
  • the fibers attach to the core particles and the binding agent fills the interstices.
  • the binding agent is then solidified by one of the means as discussed above, in such a manner that the prion complexing agent is accessible on the outer surface.
  • An example of such a system which uses iron oxide as the core particles, cellulose fibers as the fibrous material and agar as the binding agent is described in U.S. Pat. No. 5,705,628, which is incorporated herein by reference.
  • the present invention also includes within its scope a composite magnetic resin which comprises magnetic particles embedded in a organic polymer matrix which either contains, or has attached thereto, sites which are selective for prions.
  • the composite may thus comprise magnetic particles embedded in a polymeric resin which contains active sites or chemicals intended to selectively absorb to prions.
  • the polymeric resin has small particles of selective absorbers bound thereto.
  • the selective absorbers may be, for example, a metal salt of phosphotungtic acid.
  • the composite magnetic particles of the present invention may be used in a method for the removal of prions from any flowable biological sample. Removal of prions from the human product is by contacting the solution to be treated with particles of a composite magnetic resin with immobilized complexing agent and separating by magnetic filtration the composite magnetic resin particles from the solution. These magnetic particles may be used once and discarded, or recycled for use in purifying other blood products. Particles can be recycled by subjecting the separated composite magnetic resin particles to regeneration using an appropriate regenerant solution, separating the regenerated composite magnetic resin particles from the regenerant solution.
  • the composite magnetic resin particles with bound prions are then selectively removed from the solution by magnetic filtration using techniques which are known in the art.
  • the composite magnetic resin particles are then recovered from the filter and the prions removed therefrom using an appropriate regenerant solution, for example an acidic solution.
  • the cleaned composite magnetic resin particles can then be recovered from the regenerant solution by magnetic filtration and the clean particles recycled for additional use.
  • Purification of biological material from a patient may be through an extracorporal treatment unit, and following treatment the purified fluid may either be stored or may be reintroduced to the patient.
  • the biological material is pumped from for example a patient into a treating unit comprising a microporous semipermeable membrane having pores of 0.01-0.8 microns, preferably 0.15-0.45 microns.
  • a treating unit comprising a microporous semipermeable membrane having pores of 0.01-0.8 microns, preferably 0.15-0.45 microns.
  • pressure variations for example from -200 to +200 mmHg, preferably from -100 to +100 mmHg
  • a penetrating fraction of the biological material e.g. the plasma
  • Spherical beads composed of a silicate derivative are used in a cylindrical metal chromatography apparatus.
  • the beads are prepared for affinity chromatography by impregnation of the beads with PTA prior to placement within the chromatography housing apparatus.
  • Beads of approximately 5mm were impregnated with 25 wt. % loading of H 3 PW I2 O 40 by the incipient wetness method.
  • the coated beads are dried in a vacuum oven to remove the excess water. Finally, these coated beads are calcined at 350° C for 1 hour in nitrogen and 4 hours in air.
  • the coated spherical beads are cooled to approximately 37 °C, placed in the chromatography column apparatus, and equilibrated with 20 mM sodium phosphate at pH 7.
  • a preparation of human plasma is added to the mixture, which is allowed to run over the column at a speed sufficient for binding of prions to the immobilized PTA. An aliquot of the purified plasma is then tested for the presence of prions using Western blot analysis.
  • Coupling of the antibody with the membrane is allowed to take place for 15 hours at 4 "degrees C. Following incubation, the membrane is rinsed in distilled water, and the unbound ⁇ -PrP Sc is collected with the first rinsing water for later use.
  • the membrane with bound ⁇ -PrP Sc is then used in a hemofiltration apparatus to remove the PrP Sc protein from human blood.
  • the ⁇ -PrP Sc -bound filter is placed into a hemofiltration device, such as that described in U.S. Pat Nos.5, 858,238, 5,855,782, and 5,851,394 each of which are incorporated herein by reference.
  • EXAMPLE 3 One method for purifying blood of a living animal involves passing the blood through an extracorporeal shunt device.
  • a shunt device is constructed for this purpose with cellulosic hollow fibers having an ID of 200 u and inner wall thickness of 30 micrometers.
  • the cartridge formed has a total tube inner surface area of 0.6 square meter.
  • the hollow fibers are perfused with a solution of an anti-PrP Sc antibody in saline buffered with borate to maintain pH 8.5. After three hours of recirculation, the cartridge is washed extensively with saline. The solution is assayed for Anti-PrP Sc antibody by immunodiffusion technique, known to the art, before and after the recirculation step in order to evaluate the degree of antibody uptake by the fiber wall. Subsequently the cartridge is dried in a nitrogen stream, placed in a plastic bag and sealed.
  • a cartridge containing 1,000 asymmetric hollow fibers having a cut-off pore-size at 500,000 Dalton MW and inner diameter of 150-200 microns is washed by pumping saline solution through the fiber walls from the outside in the "reverse" ultrafiltration mode.
  • EXAMPLE 4 Super-paramagnetic polystyrene beads containing magnetite (average diameter 0.8 .mu.m, 67%) magnetic content—Sigma Chemical Co.) are coated overnight at room temperature with a monoclonal antibody raised native PrP Sc .
  • the resultant antibody-coated beads are placed into an apparatus comprised of a container such as a syringe body containing a support matrix surrounded by a helically wound copper wire coil, which is connected a suitable supply of alternating electric current via suitable switch means.

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Abstract

Cette invention se rapporte à des dispositifs tels que des colonnes à flux traversant, à des substrats tels que des perles polymères sphériques et à des procédés d'utilisation de ceux-ci pour éliminer les prions de n'importe quel échantillon liquide. A cet effet, une surface d'un substrat est recouverte d'un agent formant complexe avec les prions, tel qu'un sel métallique (par exemple sodium) d'acide phosphotungstique. Le sang ou le plasma traversant une colonne contenant de telles perles recouvertes de l'agent formant complexe avec les prions sont ainsi rendus exempts de tout prion. Lesdites perles peuvent être constituées par un noyau ferromagnétique visant à faciliter l'élimination des perles de l'échantillon.
PCT/US1999/030167 1999-01-20 1999-12-17 Elimination des prions du sang, du plasma et d'autres liquides WO2000043782A2 (fr)

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KR1020017009089A KR20010089612A (ko) 1999-01-20 1999-12-17 혈액, 혈장 및 다른 액체로부터 프리온의 제거
AU25898/00A AU768032B2 (en) 1999-01-20 1999-12-17 Removal of prions from blood, plasma and other liquids
IL14409799A IL144097A0 (en) 1999-01-20 1999-12-17 Removal of prions from blood, plasma and other liquids
CA002363846A CA2363846A1 (fr) 1999-01-20 1999-12-17 Elimination des prions du sang, du plasma et d'autres liquides
BR9916932-0A BR9916932A (pt) 1999-01-20 1999-12-17 Método e dispositivo para remover priÈnios de umaamostra de sangue, plasma e outros lìquidos
JP2000595152A JP2002539081A (ja) 1999-01-20 1999-12-17 血液と血漿およびその他の液体に含まれるプリオンの除去
AT99968494T ATE299268T1 (de) 1999-01-20 1999-12-17 Entfernung von prionen aus blut, plasma und anderen flüssigkeiten
DE69926081T DE69926081T2 (de) 1999-01-20 1999-12-17 Entfernung von prionen aus blut, plasma und anderen flüssigkeiten
EP99968494A EP1145013B1 (fr) 1999-01-20 1999-12-17 Elimination des prions du sang, du plasma et d'autres liquides

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023425A1 (fr) * 1999-09-28 2001-04-05 Universität Zürich Facteurs ayant une activite de liaison au prion dans du serum ou du plasma et agents permettant de detecter l'encephalopathie spongiforme transmissible
WO2002035238A1 (fr) * 2000-10-27 2002-05-02 The Regents Of The University Of California Procede permettant de determiner une souche de prions
DE10061200A1 (de) * 2000-12-08 2002-06-27 Niels Wedemeyer Verfahren und Kit zur Diagnose spongiformer Encephalopathien
WO2002063306A2 (fr) * 2001-01-16 2002-08-15 Biotransplant, Inc. Utilisation de microparticules de haute densite dans l'elimination d'agents pathogenes
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US7151000B2 (en) 1999-03-05 2006-12-19 The Regents Of The University Of California Method of concentrating proteins from serum
WO2001023425A1 (fr) * 1999-09-28 2001-04-05 Universität Zürich Facteurs ayant une activite de liaison au prion dans du serum ou du plasma et agents permettant de detecter l'encephalopathie spongiforme transmissible
WO2002035238A1 (fr) * 2000-10-27 2002-05-02 The Regents Of The University Of California Procede permettant de determiner une souche de prions
DE10061200A1 (de) * 2000-12-08 2002-06-27 Niels Wedemeyer Verfahren und Kit zur Diagnose spongiformer Encephalopathien
WO2002063306A2 (fr) * 2001-01-16 2002-08-15 Biotransplant, Inc. Utilisation de microparticules de haute densite dans l'elimination d'agents pathogenes
WO2002063306A3 (fr) * 2001-01-16 2003-11-20 Biotransplant Inc Utilisation de microparticules de haute densite dans l'elimination d'agents pathogenes
US6613505B2 (en) 2001-04-12 2003-09-02 Bioresource International, Inc. Composition and method for destruction of infetious prion proteins
WO2002088749A1 (fr) * 2001-04-25 2002-11-07 Pa Consulting Services Limited Test diagnostique ameliore pour analyse de sang
US7659076B2 (en) * 2002-02-28 2010-02-09 Microsens Biophage Limited Binding of pathological forms of prion proteins
US8658374B2 (en) 2002-02-28 2014-02-25 Microsens Biphage Limited Binding of aggregated forms of proteins
US8460885B2 (en) 2002-02-28 2013-06-11 Microsens Biophage Limited Binding of pathological forms of prion proteins
US7201901B2 (en) 2002-05-23 2007-04-10 Ortho-Clinical Diagnostics, Inc. Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration
US7252720B2 (en) 2002-06-18 2007-08-07 Common Services Agency Removal of prion infectivity
WO2003105911A1 (fr) * 2002-06-18 2003-12-24 Common Services Agency Elimination de l'infectiosite aux prions
DE102004040119A1 (de) * 2004-08-18 2006-04-27 Heinrich-Heine-Universität Düsseldorf Mittel zur Therapie und Prävention von Prionenerkrankungen
WO2008083236A1 (fr) * 2006-12-29 2008-07-10 Texas Tech University Procédé orthogonal pour l'élimination d'agents d'encéphalopathie spongiformes transmissibles à partir de fluides biologiques
ITRM20080602A1 (it) * 2008-11-07 2010-05-08 Cbm Scrl Consorzio Per Il Ct Di Medicina Molec Nanoparticelle di oro rivestite con polielettroliti e loro uso come medicamento per il trattamento di malattie neurodegenerative causate da aggregati proteici
WO2010052665A2 (fr) * 2008-11-07 2010-05-14 Consorzio Per Il Centro Di Biomedicina Molecolare Scrl Nanoparticules d'or revêtues de polyélectrolytes et leur utilisation en tant que médicament pour le traitement de maladies neurodégénératives provoquées par des agrégats protéiques
WO2010052665A3 (fr) * 2008-11-07 2010-07-01 Consorzio Per Il Centro Di Biomedicina Molecolare Scrl Nanoparticules d'or revêtues de polyélectrolytes et leur utilisation en tant que médicament pour le traitement de maladies neurodégénératives provoquées par des agrégats protéiques
US9446003B2 (en) 2009-04-15 2016-09-20 Abraxis Bioscience, Llc Prion free nanoparticle compositions and methods of making thereof
US10206887B2 (en) 2009-04-15 2019-02-19 Abraxis Bioscience, Llc Prion free nanoparticle compositions and methods of making thereof

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CN1357109A (zh) 2002-07-03
AU2589800A (en) 2000-08-07
CN1182399C (zh) 2004-12-29
US6916419B2 (en) 2005-07-12
US6221614B1 (en) 2001-04-24
DE69926081T2 (de) 2006-04-20
US20010005578A1 (en) 2001-06-28
KR20010089612A (ko) 2001-10-06
AU768032B2 (en) 2003-11-27
ES2242447T3 (es) 2005-11-01
CA2363846A1 (fr) 2000-07-27
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IL144097A0 (en) 2002-05-23
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